We, the present inventors, previously have found that a water-soluble chitin-oligomer or chitin-oligosaccharide selected from di-N-acetyl-chitobiose, tri-N-acetyl-chitotriose, tetra-N-acetyl-chitotetraose, penta-N-acetyl-chitopentaose, hexa-N-acetyl-chitohexaose and hepta-N-acetyl-chitoheptaose is useful as the antibacterial and antifungal agents and also as the antitumor agent owing to the immunopotentiating activity of these water-soluble chitin-oligomers when administered intraperitoneally, intravenously or orally to man (see Japanese patent applications Nos. 252761/84 and 50618/85; U.S. patent application Ser. No. 800,774 and European patent application No. 85 308687).
These chitin-oligomers are represented by a general formula ##STR1## wherein m is zero for di-N-acetyl-chitobiose, m is 1 for tri-N-acetyl-chitotriose, m is 2 for tetra-N-acetylchitotetraose, m is 3 for penta-N-acetyl-chitopentaose, m is 4 for hexa-N-acetyl-chitohexaose and m is 5 for hepta-N-acetyl-chitoheptaose. Amongst these chitin-oligomers, tetra-N-acetyl-chitotetraose, penta-N-acetyl-chitopentaose and hexa-N-acetyl-chitohexaose are most preferred owing to their higher immunopotentiating activities than those of the other chitin-oligomers. These water-soluble chitin-oligomers may be produced by partial hydrolysis of chitin, along with a larger quantity of N-acetylglucosamine which is the monomeric unit of the chitin. Chitin is readily available as chitin materials occur in large quantities in nature, for example, in the outer shells of Crustacea such as crabs and lobsters, and also in the shells of insects and the cell walls of various kinds of microorganisms.
It is known that chitin is insoluble in water, organic solvents and aqueous alkali metal hydroxide solutions and that chitin is less wettable by water and is somewhat resistant to alkalis and diluted acids. It is also known that when chitin has been completely hydrolyzed by reacting with a concentrated mineral acid such as concentrated hydrochloric acid for a prolonged reaction time, it can ultimately be converted into glucosamine and acetic acid, with formation of N-acetylglucosamine as one of the intermediate products. Besides, it is known that chitin is substantially not hydrolyzable with a diluted aqueous hydrochloric acid in a reasonable reaction time.
For the production of the above-mentioned chitinoligomers from the chitin, a method is known, which comprises adding slowly and gradually a finely ground chitin in small portions into a volume of a concentrated hydrochloric acid in such a way that each small portion of the chitin as added is each time dispersed well in the volume of the concentrated hydrochloric acid used, until the total quantity of the finely ground chitin used can be dispersed in the hydrochloric acid, with requiring a long time for the formation of such dispersion containing the total quantity of the fine chitin particles dispersed in the acid, and then effecting the hydrolysis of the chitin in the resulting dispersion (the reaction mixture of the chitin with the concentrated hydrochloric acid) at an elevated temperature of about 35.degree. C. to 40.degree. C., and subsequently neutralizing the hydrolyzed reaction mixture with aqueous sodium hydroxide, followed by filtering the neutralized reaction mixture, de-salting the resulting filtrate solution containing the soluble hydrolysis products of chitin, recovering the chitin-oligomers from the de-salted filtrate solution and separating chromatographically the chitin-oligomers from each other, on a column of a gelfiltration agent such as Sephadex LH 20 (a product of Pharmacia Fine Chemical Co., Sweden) (see the "Journal of the Chemical Society" 1970, pp. 1654-1655). We have after-tested this known method and have found that with this known method, it is required that the finely ground chitin of the aforesaid nature should be carefully and slowly admixed in small portions with the concentrated hydrochloric acid, with needing a long time for mixing well the fine chitin particles with the acid, as, otherwise, undissolved small lumps of the chitin powder would remain without being wetted by and mixed with the hydrochloric acid agent. Even if a mixture of the chitin powder with the concentrated hydrochloric acid can occasionally have been obtained through severe mixing of the chitin powder with a proper volume of the concentrated hydrochloric acid, the mixture so obtained is usually in the form of a pretty stiff dough-like mass which is not agitatable by means of a mechanical stirrer. In addition, if the volume of the concentrated hydrochloric acid used per a given quantity of the chitin powder is increased excessively for the purpose of enabling the chitin powder to be well mixed with and dispersed in the hydrochloric acid and give a more fluid and agitatable dispersion or mixture, the hydrolysis of chitin can proceed excessively to give increased yields of glucosamine and N-acetylglucosamine which are not desired in the present invention, and also give decreased yields of the tetra-N-acetyl-chitotetraose, penta-N-chitopentaose and hexa-N-acetyl-chitohexaose which are most desired to be recovered in the present invention.
According to the known method described in the above-mentioned "Journal of the Chemical Society" 1970, pp. 1654-1655, an experiment was made, where finely ground chitin (100 g) was slowly added to with stirring and then hydrolyzed with 200 ml of a concentrated hydrochloric acid for totally 3 hours, the hydrolyzed reaction mixture was neutralized with 50% aqueous sodium hydroxide and filtered, followed by de-salting the neutral filtrate solution (300 ml) on a column of Sephadex G-25 and freeze-drying the resulting de-salted fractions of the effluent to yield of a mixture of N-acetylglucosamine and its oligosaccharides with degrees of polymerisation of 2 to 6, namely the di-N-acetyl-chitobiose, tri-N-acetyl-chitotriose, tetra-N-acetylchitotetraose, penta-N-acetylchitopentaose and hexa-N-acetyl-chitohexaose (hereinafter sometime abbreviated as NACOS-2-, NACOS-3, NACOS-4, NACOS-5 and NACOS-6, respectively), and where said mixture of N-acetylglucosamine and NACOS-2 to NACOS-6 was obtained only in a poor yield of 6.5 g from 100 g of the chitin material charged. In this experiment, said mixture (6.5 g) of N-acetylglucosamine and NACOS-2 to NACOS-6 was then chromatographed on a column of Sephadex LH 20 to give 0.13 g of NACOS-6, 0.3 g of NACOS-5, 0.47 g of NACOS-4, 0.81 g of NACOS-3, 0.78 g of NACOS-2 and 0.70 g of N-acetylglucosamine (NACOS), respectively. Thus, according to the aforesaid known method, the total yields of hexa-N-acetyl-chitohexaose (NACOS-6), penta-N-acetyl-chitopentaose (NACOS-5) and tetra-N-acetyl-chitotetraose (NACOS-4) recovered from chitin were considerably smaller than the total yields of the lower chitin-oligomers such as di-N-acetyl-chitobiose (NACOS-2) and tri-N-acetyl-chitotriose (NACOS-3) and were difficult to be further increased.
According to the above-mentioned known method, it is required that the powder (100 g) of the starting chitin material is so added in small portions slowly and gradually into the volume (200 ml) of the concentrated hydrochloric acid used, that the resulting mixture is always kept to be in the form of a uniform liquid dispersion which is agitatable by means of a mechanical stirrer, and due to this, a considerably long time must be taken to complete even the mixing of the total quantity of the chitin powder used, with the concentrated hydrochloric acid agent. Thus, then it is considered that the first portions of the whole chitin powder which have been added at first into the concentrated hydrochloric acid at the early stage of mixing the chitin powder with the hydrochloric acid can be exposed to and reacted with the excessive quantity of the hydrogen chloride present in the resulting dispersion (the reaction mixture) for an improperly prolonged time and hence can be hydrolyzed to such excessive extents as to produce predominantly N-acetylglucosamine and even glucosamine as the ultimate hydrolysis products of chitin, whereas the latter portions of the whole chitin powder which are added later into the mixture at the later stage of mixing the chitin powder with the hydrochloric acid are not hydrolyzed to such excessive extents as above. For these reasons, it is considered that the above-mentioned known method is difficult to provide better yields of the desired tetra-N-acetyl-chitotetraose, penta-N-acetyl-chitopentaose and hexa-N-acetyl-chitohexaose recovered. When an increased volume of a diluted hydrochloric acid has been mixed with the chitin powder with an intention of giving a more fluid and agitatable mixture, the hydrolysis of chitin cannot proceed to a substantial extent in a reasonable reaction time.